CN110157647A - It is a kind of to alleviate anxiety, Lactobacillus brevis of improvement sleep and application thereof - Google Patents
It is a kind of to alleviate anxiety, Lactobacillus brevis of improvement sleep and application thereof Download PDFInfo
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- CN110157647A CN110157647A CN201910463154.7A CN201910463154A CN110157647A CN 110157647 A CN110157647 A CN 110157647A CN 201910463154 A CN201910463154 A CN 201910463154A CN 110157647 A CN110157647 A CN 110157647A
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- lactobacillus brevis
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- lactobacillus
- sleep
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- A—HUMAN NECESSITIES
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- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C11/00—Milk substitutes, e.g. coffee whitener compositions
- A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
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- A—HUMAN NECESSITIES
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- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
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- A23L19/20—Products from fruits or vegetables; Preparation or treatment thereof by pickling, e.g. sauerkraut or pickles
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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Abstract
The invention discloses one kind can alleviate anxiety, the Lactobacillus brevis for improving sleep and application thereof, belongs to microorganisms technical field.There is Lactobacillus brevis GDMCC No.60605 of the invention excellent simulated gastrointestinal tract survival, cell adherence and GABA to generate ability.Spacious field experiment in can dramatically increase mouse enter middle section frequency, extend mouse spacious field middle section time without influence the total move distance of mouse；The number that mouse enters open arms can be dramatically increased in elevated plus-maze test；It is able to extend the sleeping time of yellow Jackets induction, shortens the Sleep latency of barbital sodium induction；Intestinal flora can be adjusted, the relative abundance of Allobaculum in mouse intestinal is dramatically increased.The Lactobacillus brevis GDMCC No.60605 is used to prepare the pharmaceutical composition and fermented food alleviated anxiety, improve sleep, has very extensive application prospect.
Description
Technical field
The present invention relates to one kind can alleviate anxiety, the Lactobacillus brevis for improving sleep and application thereof, belongs to microbial technique
Field.
Background technique
Insomnia (insomnia) is to fall asleep and sleep sleep quality caused by maintaining difficulty or the time is not achieved normally
A kind of subjective experience of psychological need is the most common sleep disordered disease.People commonly use some clinical symptoms to diagnose and lose
It sleeps, including: difficulty falling asleep, night easily wake up and difficulty falling asleep, next day early awakening, sleep without recovery effects etc. again.According to
Disease incidence and population statistics show that insomnia is generally existing one of the pain of today's society people.China is at least
35% people's insomnia, 17% people's insomnia is quite serious, and sleeps and be confined to the ratio of the people of " shallow sleep ", up to
77.3%.Insomnia will lead to and can not regain one's vigor, thus body is tired, apathetic, drowsiness, aprosexia, thinking difficulty,
It is slow in reacting, depressed, impatient, furthermore it may have a negative impact to cardiac function, immune function and blood glucose-control.
Insomnia summation anxiety, depression are accompanied, and 40% insomniac has one or more of phrenoblabias, wherein anxiety
Obstacle accounts for 24%.There are moderate correlations with anxiety for insomnia.Wherein in the patient of insomnia and anxiety comorbidity, anxiety
The case where obstacle is prior to having a sleepless night accounts for 73%, and has a sleepless night and account for 69% prior to anxiety.Therefore say that insomnia is the common disease of anxiety attack
One of shape and the principal element of anxiety morbidity, in turn, anxiety is also possible to the risk factor of chronic insomnia.
Western medicine anxiety and vigilance disorders rapid-action, that effect is strong, relatively common drug are clinically often selected now
Mainly contain barbiturates, non-benzene phenodiazineClass and benzene phenodiazineClass several types.Coke can be effectively relieved in these drugs
Consider, increases the time of sleep, reduce the incubation period of sleep, sleep quality is improved, but can but inhibit central nervous system
System for a long time using patient will be allowed to produce dependence and tolerance, such as once is discontinued that will to generate withrawal symptom etc. each
Kind adverse reaction.In view of various problems existing for traditional therapy, the new intervention of one kind is found for anxiety and insomnia or is controlled
Treatment method seems very necessary.
γ-aminobutyric acid (GABA) is a kind of important neurotransmitter, Japanese health ministry, European Food Safety Authority (EFSA)
Recognize that the GABA of lactobacillus-fermented production is natural additive for foodstuff with Food and Drug Adminstration of the US (FDA).2009, China
It is new resource food that the Ministry of Public Health, which ratifies such GABA,.Studies have shown that GABA can prevent nerve excessively nervous and emerging with calm nerves
It puts forth energy, can preferably alleviate stress, adjust mood, restore physically and mentally healthy, while can be shortened time for falling asleep and extending deep sleep
There is certain remission effect in time to insomnia.Typically now think, the GABA of meal supplement may be by improving the micro- life of enteron aisle
The approach such as state and vagus nerve access adjust brain function, and then alleviate anxiety, improve sleep.
At the same time, Long-Term Scientific Study the result shows that, enteric microorganism passes through directly contact, shla molecule and host
Biological interaction, own metabolism provide multiple beneficial function for host.Between gastrointestinal tract and central nervous system (CNS)
The communication path between intestinal microbiota and the neural circuit (including CNS) of host is also supported in intestines-brain axis two-way communication.It is more next
More evidences shows that intestinal microbiota can adjust enteron aisle and cerebral function, the mood including the mankind, cognitive function and row
For.Wherein lactic acid bacteria is the important component of enteric microorganism, and the lactic acid bacteria of many kinds also has the ability for producing GABA.?
In this case, giving full play to lactic acid bacteria can produce the advantage of GABA, and bacterial strain itself and the GABA generated is made to form compound effect
Answer, collective effect in body come alleviate anxiety, improve sleep.
But produce that the lactic acid bacteria of GABA is different in vivo surely to be had the effect of alleviating anxiety, improve sleep, because having
Many external lactic acid bacterias for producing GABA can not survive when passing through gastrointestinal tract or cell adherence ability is poor, can not be in intestinal colonisation
Play its beneficial effect.And have some lactic acid bacterias strong with stronger gastrointestinal tract tolerance, cell adherence ability, do not have but
Higher GABA generates ability, it is difficult to play preferable beneficial effect.In addition, probiotics has the acid and cholate of tolerance gastrointestinal tract
Environment and to colonize in enteron aisle be prerequisite condition that probiotics functions in vivo.Therefore, seek a kind of with excellent
Gastrointestinal tract survival ability, cell adherence ability and GABA generate the lactic acid bacteria of ability and be a problem to be solved.
Currently, some patent documents are related to alleviating anxiety, improving sleep health food and preparation method thereof, such as patent
CN103141855A discloses a kind of by γ-aminobutyric acid, serotonin, vitamin B60With the improvement sleep matter of Schisandra chinensis compounding
Health care product of amount and preparation method thereof；Patent CN103976351A discloses a kind of lactic acid bacteria progress liquid deep layer fermenting production
GABA, preparation while the method with the fermentation liquid of strengthen immunity and improvement sleep function；CN107427539A discloses one
Kind includes at least bifidobacterium bifidum W23 and can control the probiotic composition of gut barrier function, can treat and prevents
It is related to the human diseases of brain-gut axis.
But above-mentioned patent is not directed to while having excellent gastrointestinal tract survival ability, cell adherence ability and GABA to produce
The lactic acid bacteria of raw ability, really can not be used to improve sleep.
Summary of the invention
The object of the present invention is to provide a kind of Lactobacillus brevis (Lactobacillus brevis) GDMCC No.60605.
It is related to a kind of Lactobacillus brevis present invention as described above, utilizes morphological feature, cultural colony and physiological and biochemical property
Etc. Microbiological Characteristics Lactobacillus brevis (Lactobacillus brevis) GDMCC No.60605 is accredited as to the lactic acid bacteria, should
For bacteria strain in Guangdong Culture Collection preservation, deposit number is GDMCC No.60605.
Lactobacillus brevis of the invention, has the property that
1, morphological feature:
Gram's staining result is gram-positive bacteria；Microscopy is in rod-short, both ends circle, single or at short chain, atrichia,
Without spore.
2, cultural colony:
Bacterium colony after cultivating 48h on MRS culture medium is generally creamy white, coarse flat, not chromogenesis.
3, physiological and biochemical property:
Arabinose, fructose, glucose, maltose, lactose, melibiose, ribose and sucrose can be utilized；Do not utilize horse chestnut
Glycosides, salicin, gossypose, sorbierite, inositol, dulcitol, rhamnose, galactolipin；Catalase, H2S is generated, urea divides
Solution, nitrate reduction etc. are feminine gender.Energy is generated with excellent simulated gastrointestinal tract survival ability, cell adherence ability and GABA
Power；Its content that the GABA in 48h fermented supernatant fluid is cultivated in MMRS culture medium is 5072 ± 280mg/L.
4, stomach-filling mouse effect:
(1) frequency for making mouse enter middle section in spacious field experiment increases 55.4%, makes mouse in spacious field center
The time in region extends 121.1%, has significant difference without influencing the total move distance of mouse compared with naive mice；
(2) number for making mouse enter open arms in elevated plus-maze test increases 69.4%, with naive mice
Compared to there is significant difference；
(3) sleeping time of yellow Jackets induction can significantly be extended, extended ratio is 149.2%, makes barbital
The Sleep latency of sodium induction shortens 31.0%；
(4) intestinal flora can be adjusted, the relative abundance of Allobaculum in mouse intestinal is dramatically increased.
A second object of the present invention is to provide the leavenings for containing the Lactobacillus brevis GDMCC No.60605.
In one embodiment, the leavening is to utilize the bacterium solution containing the Lactobacillus brevis GDMCC No.60605
Obtained pulvis is prepared, it contains 106The active Lactobacillus brevis GDMCC No.60605 of CFU/g or more.
In one embodiment, the pulvis is to pass through the bacterium solution containing the Lactobacillus brevis GDMCC No.60605
Normal freeze-drying technique or other techniques prepare obtained.
In one embodiment, the leavening is obtained by following preparation steps:
A, the preparation of culture medium: use in terms of the total weight of medium 87.7% water by 10% enzyme hydrolysis skimmed milk,
0.5% glucose, 1.5% tryptone and 0.3% yeast extract dissolve, and then adjusting its pH is 6.8, obtain so described
Culture medium；
B, it protectant preparation: is mixed with to obtain containing 100g/L skimmed milk power, 30mL/ using water and protective agent raw material
L glycerol, 100g/L maltodextrin, 150g/L trehalose, 10g/L L-sodium protective agent；
C, Lactobacillus brevis GDMCC No.60605 strain is connect according to 2~4% inoculum concentration of poidometer of the culture medium
Then kind is cultivated into the culture medium for sterilizing 8~12min at 110~120 DEG C of temperature under conditions of 37 DEG C of temperature
18h is cleaned 2~4 times with pH7.2 phosphate buffer, reaches concentration 10 with the protective agent resuspension10CFU/mL；Then, it allows
Suspension preculture 60min under conditions of 37 DEG C of temperature, then be freeze-dried to obtain the leavening.
In one embodiment, the leavening also contains the microorganism that can be used for food.Optionally, also containing acidproof
One of lactobacillus, lactobacillus plantarum, lactobacillus bulgaricus are two or more.
Third object of the present invention is to provide the applications of the Lactobacillus brevis GDMCC No.60605.
Optionally, the application is to be used to prepare fermented food.The fermented food is using Lactobacillus brevis of the invention
What GDMCC No.60605 or leavening containing Lactobacillus brevis GDMCC No.60605 strain produced.
In one embodiment, Lactobacillus brevis GDMCC No.60605 viable bacteria is contained in the fermented food.
In one embodiment, the fermented food is dairy products, bean product or fruit and vegetable product.Optionally, described
Dairy products be milk, sour cream or cheese.Optionally, the bean product are soymilk, fermented soya bean or beans sauce.Optionally, described
Fruit and vegetable product be cucumber, carrot, beet, celery or cabbage product.
Optionally, the application is to be used to prepare the pharmaceutical composition with sleep function is improved.
In one embodiment, described pharmaceutical composition is by Lactobacillus brevis GDMCC No.60605 microbial inoculum and in pharmacy
Upper acceptable carrier composition.
In one embodiment, pharmaceutically acceptable carrier is one or more selected from pharmaceutically usually used
Filler, adhesive, wetting agent, disintegrating agent, lubricant or corrigent carrier.Optionally, the pharmaceutical composition is
Granule, capsule, tablet, pill or oral liquid formulation.
Beneficial effect and advantage of the present invention are as follows:
Lactobacillus brevis GDMCC No.60605 of the invention, with excellent simulated gastrointestinal tract survival, cell adherence and
GABA generates ability, and (content that the GABA in 48h fermented supernatant fluid is cultivated in MMRS culture medium is 5072 ± 280mg/L.).
The frequency that mouse enters middle section can be dramatically increased in spacious field experiment, extend mouse in the time of spacious field middle section and
The total move distance of mouse is not influenced；The number that mouse enters open arms can be dramatically increased in elevated plus-maze test；It can
Extend the sleeping time of yellow Jackets induction, shorten the Sleep latency of barbital sodium induction；Intestinal flora can be adjusted, is shown
Write the relative abundance for increasing Allobaculum in mouse intestinal.The Lactobacillus brevis GDMCC No.60605 is used to prepare slow
The pharmaceutical composition and fermented food for solving anxiety, improving sleep, have very extensive application prospect.
Biomaterial preservation
Lactobacillus brevis (Lactobacillus brevis), classification naming be Lactobacillus brevis, with
It is preserved in Guangdong Province's Culture Collection on March 12nd, 2019, preservation address is the compound of Xianlie Middle Road, Guangzhou City 100
5 building, No. 59 building, deposit number are GDMCC No.60605.
Detailed description of the invention
Fig. 1 is influence of the Lactobacillus brevis GDMCC No.60605 to the total move distance of mouse in spacious field experiment；
Fig. 2 be spacious field experiment in Lactobacillus brevis GDMCC No.60605 to mouse the middle section residence time influence；
Fig. 3 is the influence that Lactobacillus brevis GDMCC No.60605 enters middle section frequency to mouse in spacious field experiment；
Fig. 4 is different disposal group mouse movement trajectory diagram comparison in spacious field experiment.
Fig. 5 be in elevated plus-maze test Lactobacillus brevis GDMCC No.60605 to mouse the open arms residence time shadow
It rings.
Fig. 6 is the shadow that Lactobacillus brevis GDMCC No.60605 enters open arms number to mouse in elevated plus-maze test
It rings.
Fig. 7 is the influence for the sleeping time experiment that Lactobacillus brevis GDMCC No.60605 induces yellow Jackets.
Fig. 8 is the influence for the Sleep latency experiment that Lactobacillus brevis GDMCC No.60605 induces barbital sodium.
Fig. 9 is influence of the Lactobacillus brevis GDMCC No.60605 to mouse intestinal flora beta diversity.
Figure 10 is the influence that Lactobacillus brevis GDMCC No.60605 changes Allobaculum relative abundance.
(note: p < 0.01 * p < 0.05, * *)
Specific embodiment
Experimental method described in following embodiments is unless otherwise specified conventional method；The reagent and biological material
Material, unless otherwise specified, commercially obtains.
The screening of 1 Lactobacillus brevis GDMCC No.60605 of embodiment
1 experimental method
Bacterial strain screening is carried out using fresh crowd's excrement and pickles water as sample.
(1) pipette samples 0.5mL is in the MRS culture medium of 5mL, and 37 DEG C of cultures 18~for 24 hours, it is enriched with.
(2) gradient dilution: the sample 0.5mL after drawing enrichment obtains 10 in 4.5mL sterile saline-1Dilution,
Then 0.5mL 10 is drawn-1Dilution obtains 10 in 4.5mL physiological saline-2Dilution successively obtains 10 by this operation-3,
10-4, 10-5, 10-6Dilution.
(3) coated plate culture: drawing 100 μ L gradient dilution liquid and be coated on MRS plate, and 10-4, 10-5, 10-6Each gradient 1
Plate；In 37 DEG C of culture 48h.
(4) scribing line separation: select have characteristic feature on spread plate according to colony shape, size, edge, transparency etc.
Bacterium colony is crossed on selective medium plate with oese picking colony, 37 DEG C of culture 48h.
(5) single colonie purified, one single colonie of picking are seeded in corresponding 5mL fluid nutrient medium, culture 18
~for 24 hours.By each strain number, the experiment such as bacterial strain identification, Gram's staining, Physiology and biochemistry is carried out.
Wherein, the formula of MRS culture medium are as follows: in 1L distilled water be added 10g peptone, 10g beef extract, 20g glucose,
5g yeast extract, 2g anhydrous sodium acetate, 0.25g manganese sulfate monohydrate, 1mL Tween 80,2.6g dipotassium hydrogen phosphate trihydrate, 0.5g seven
Water magnesium sulfate, 2g dibasic ammonium citrate, pH 6.2~6.5.
2 experimental results
PCR amplification 16S rDNA carried out to isolated bacterial strain, PCR product send to Hua Da gene sequencing Co., Ltd into
Row sequencing, GDMCC No.60605 qualification result are Lactobacillus brevis (Lactobacillus brevis), it is characterized in that:
(1), morphological feature:
Gram's staining result is gram-positive bacteria；Microscopy is in rod-short, both ends circle, single or at short chain, atrichia,
Without spore.
(2), cultural colony:
Bacterium colony after cultivating 48h on MRS culture medium is generally creamy white, coarse flat.
(3), physiological and biochemical property:
Arabinose, fructose, glucose, maltose, lactose, melibiose, ribose and sucrose can be utilized；Do not utilize horse chestnut
Glycosides, salicin, gossypose, sorbierite, inositol, dulcitol, rhamnose, galactolipin；Catalase, H2S is generated, urea divides
Solution, nitrate reduction etc. are feminine gender.
2 Lactobacillus brevis GDMCC No.60605 of embodiment produces GABA capability study
1 test method
The activation of 1.1 Lactobacillus brevis GDMCC No.60605
Lactobacillus brevis GDMCC No.60605 is seeded in MRS culture medium with 2% inoculum concentration, at 37 DEG C, culture
18h, the Lactobacillus brevis GDMCC No.60605 after being activated；
The preparation of 1.2 fermentation liquids
It is activated described in 1.1 and takes 2% to be inoculated into addition 1.5%L- sodium glutamate (L-MSG) in bacterium solution twice
In MMRS culture medium 37 DEG C of culture 48h to get arrive fermentation liquid to be measured.
MMRS culture medium prescription is (g/L): 1.5% L-sodium conduct is added on the basis of MRS culture medium
The precursor substance of GABA synthesis.
The measurement of GABA in 1.3 fermentation liquids
Use the content of the GABA in HPLC measurement fermented supernatant fluid.Analysis condition is repaired according to national standard QBT 4587-2013
Change.Chromatographic condition are as follows: chromatographic column: Hypersil GOLD chromatographic column (100mm × 2.1mm)；30 DEG C of column temperature；Sample volume: 5 μ
L；Mobile phase A is 20mmol/L sodium acetate aqueous solution, and Mobile phase B is V (40mmol/L sodium acetate aqueous solution): V (acetonitrile)=1:
1；Flow velocity 0.2mL/min；Detection wavelength 338nm.Gradient are as follows: 0~6min, B rise to 50% by 30%；6~11min, B by
50% rises to 60%；11~12min, B rise to 100% by 60% and keep 3min；15~16min, B are down to 30% by 100%；
16~20min, 30%B keep 4min.
Sample pretreatment: supernatant is diluted certain multiple by sample 1mL, 8000r/min the centrifugation 5min after taking fermentation,
Through 0.22 μm of filtering with microporous membrane, derivatization reaction can be used to.
Sample derivatization: (1) preparation of 0.4mol/L borate buffer: accurately weighing 2.47g boric acid, adds water about 80mL,
PH is adjusted to 10.2 with NaOH, is settled to 100mL with water.(2) preparation of derivative reagent: 0.1g o-phthalaldehyde (o- is weighed
Phthalaldehyde, OPA), it is dissolved with 1mL acetonitrile, 130 μ L mercaptoethanols is then added, are settled to 100mL with water.(3) sample
Product column front derivation: drawing each 10 μ L of sample after derivative reagent and pretreatment, reacts sample introduction after 90s at room temperature after mixing.
2 experimental results
It is found after content using the GABA cultivated in HPLC measurement MMRS culture medium in 48h fermented supernatant fluid, short cream bar
Bacterium GDMCC No.60605 has higher GABA generation horizontal in vitro, in fermented supernatant fluid GABA content be 5072 ±
280mg/L。
3 Lactobacillus brevis GDMCC No.60605 simulated gastrointestinal tract survival ability of embodiment is probed into
1 test method
(1) configuration of simulate the gastric juice: pepsin is dissolved in the physiological saline of sterilizing, and (0.9%w/v, hydrochloric acid tune pH is extremely
3.0) in, make final concentration of 3g/L.It is filtered with 0.22 μm of sterilised membrane filter, it is ready-to-use.
(2) configuration of simulated intestinal fluid: trypsase is dissolved in the physiological saline of sterilizing, and (0.9%w/v, NaOH tune pH are extremely
8.0) in, make final concentration of 1g/L, and cholate is added to make final concentration of 0.3%.It is filtered with 0.22 μm of sterilised membrane filter, now with existing
With.
(3) it is tested after Lactobacillus brevis GDMCC No.60605 continuously being activated three generations (each 18h).Measurement is continuous living
Change the bacterial strain of three generations in A600Under OD value, by calculate find out OD 5 needed for bacterium solution amount.
(4) bacterium solution of OD 5 is centrifuged 10min with 8000 × g, abandons supernatant, is resuspended in 1mL simulate the gastric juice, 37 DEG C of cultures
Viable plate count is carried out after 3h.
(5) simulate the gastric juice is taken treated 8000 × g of bacterium solution centrifugation 10min, abandon supernatant, be resuspended in isometric simulation intestines
In liquid, viable plate count is carried out after 37 DEG C of culture 4h.
Wherein, survival rate (%)=(original in viable count/bacterium solution after tolerance simulate the gastric juice in bacterium solution after being resistant to gastric juice
Beginning viable count) × 100%.Survival rate (%)=(viable count/tolerance mould after tolerance simulated intestinal fluid in bacterium solution after being resistant to intestinal juice
Viable count after quasi- gastric juice in bacterium solution) × 100%.Be resistant to gastro-intestinal Fluid after survival rate (%)=(tolerance simulated intestinal fluid after bacterium solution
In viable count/bacterium solution in original viable count) × 100%.
2 experimental results
Tolerance of the 1 Lactobacillus brevis GDMCC No.60605 of table in simulated gastrointestinal tract
By count plate, it is found that survival rate of the Lactobacillus brevis GDMCC No.60605 after simulate the gastric juice culture 3h is
92.08 ± 3.09%, the survival rate after 4h is cultivated in simulated intestinal fluid is 18.30 ± 0.97%, always depositing in simulated gastrointestinal tract
Motility rate is 16.85 ± 1.06%.Show that Lactobacillus brevis GDMCC No.60605 has excellent gastrointestinal tract tolerance, it can be in meals
Tonic reaches enteron aisle after filling, and plays prebiotic function.
4 Lactobacillus brevis GDMCC No.60605 of embodiment probes into the external adhesive capacity of HT-29 cell
1 test method
Normal HT-29 is cultivated into cell and is passed on, Lactobacillus brevis GDMCC No.60605 continuously activated for 3 generations.Collect culture
Good cell, is counted with blood counting chamber, and is resuspended with 1640 culture mediums (adding dual anti-, fetal calf serum) to 2 × 105A/mL.
The coverslip through pickling and sterilizing is added in 6 orifice plates.Inoculating cell suspension 2mL, it is adherent after coverslip (about 12h) after cell
Sterile PBS is washed 3 times.Taking a certain amount of bacterium solution, thalline were collected by centrifugation, and bacterium is collected by centrifugation again after cleaning thallus 1 time with sterile PBS
Body is resuspended to bacteria concentration with 1640 culture mediums (not adding dual anti-, fetal calf serum) as 108CFU/mL.6 orifice plates are added in every hole
1640 culture medium bacteria suspension of 2mL is incubated for 2h.After incubation, culture solution is discarded, sterile PBS is washed 3~6 times.Cleaning terminates
Afterwards, the fixed 1h of 2mL methanol room temperature is added in every hole.After fixation, methanol, gram (crystal violet) dyeing, micro- are discarded
Under the microscope.
2 experimental results
By the external adherence test of HT-29 cell, micro- sem observation discovery Lactobacillus brevis GDMCC No.60605's is thin
It is 28.67 ± 4.73/cell that born of the same parents, which adhere to number,.And the preferable cell adhesion ability of Lactobacillus brevis GDMCC No.60605 is advantageous
In it in intestinal colonisation, GABA is generated, the beneficial functions of itself are played.
The preparation of 5 Lactobacillus brevis GDMCC No.60605 freeze-dried vaccine powder of embodiment
Bacterial strain after activation is seeded in MRS culture medium with 2% inoculum concentration, at 37 DEG C, after cultivating 18h, 8000
Supernatant is discarded after × g centrifugation 10min, collects thallus after washing 3 times with sterile saline, obtained above-mentioned thallus will be collected and used
Protective agent is resuspended, and freeze-drying obtains freeze-dried vaccine powder, and the protective agent can be skimmed milk power, trehalose or sucrose etc.；
6 Lactobacillus brevis GDMCC No.60605 of embodiment alleviates anxiolytic effect evaluation
1 zoopery design
The 6 week old health male ICR mouses 30 for taking 20~25g, are randomly divided into 3 groups: blank control group, positive control
Group, Lactobacillus brevis GDMCC No.60605 treatment group.The daily stomach-filling sterile saline of blank control group；Lactobacillus brevis GDMCC
No.60605 treatment group feeds the concentration 3 × 10 of the preparation of this specification embodiment 5 daily9CFU/mL Lactobacillus brevis GDMCC
No.60605 degreasing emulsion suspension liquid, the diazepam solution of positive controls stomach-filling 3mg/kg.bw.Mouse phase is orally given once a day
The tested material of dosage is answered, intragastric administration on mice amount is 20mL/kg.bw, continues 30 days.
The experiment of 1.1 spacious fields
Spacious field experiment is carried out after last stomach-filling 30min, and the testing time is 8:00~15:00, mouse is put into before test
It is covered in the big box of padding, is allowed to freely explore 5min.When experiment starts, mouse is put into rapidly to spacious field center, uses video
Track and record the activity condition in mouse 10min.Spacious field is wiped with wet cloth after test, excrement is removed, after being cleaned with dry cloth
The test of next mouse is carried out again.It is spacious to move total distance in 10min, into middle section number, residence time as index
Field experimental result is listed in attached drawing 1~4.
1.2 elevated plus-maze test
Spacious field experiment is carried out after last stomach-filling 30min, the testing time is 8:00~15:00.Mouse elevated plus-maze
It is made of the central platform of four two opposite open arms, two opposite closure arms and a connection arms.Central platform connects
Connect two open arms and two closure arms.Labyrinth is integrally fastened on bracket, make labyrinth bottom plate away from experiment room floor 50cm at.It is real
It tests interior to illuminate with portable lamp, keep quite.Mouse is put into animal box before test, is allowed to move freely 5min.Experiment is opened
When the beginning, mouse is placed at central platform, mouse head is allowed to free exploration towards open arms.Mouse in 5min is recorded respectively to enter
Open arms number (open arms entries, OE) closes arm number (close arms entries, CE), into the open arms time
(time spent in open arms, OT), into closing the arm time (time spent in close arms, CT).To enter
Open arms number and enter the percentage (OE%) of arm total degree and run duration and the percentage of total time (OT%) are made in open arms
For the index for evaluating mouse anxiety state.Alcohol wipe labyrinth is used after test every time, removes excreta, then carry out next time
Test.Every mouse only uses once, if falling down overhead, terminates to test.Elevated plus-maze test result be listed in attached drawing 5~
In 6.
2 experimental results
2.1 spacious field experimental results are as shown in Figures 1 to 3 to be compared with blank group, and Lactobacillus brevis GDMCC No.60605 is in spacious field
The frequency for making mouse enter middle section in experiment increases 55.4%, extends mouse in the time of spacious field middle section
121.1%, there is significant difference compared with naive mice, but above-mentioned difference is not due to the difference of mouse movement total distance
It is caused.Mouse motion profile figure is also this it appears that mouse is explored after stomach-filling Lactobacillus brevis GDMCC No.60605 in Fig. 4
The increase of behavior, anxiety symptom mitigate.
2.2 elevated plus-maze test results
Mouse can live with inquiry in open arms again out of curiosity since thermophilic dark property can tend to the activity in closing arm
Dynamic, when facing novel stimulus, animal generates the impulsion probed into and fear simultaneously, this has resulted in the row that conflicts probed into and avoided
For to generate anxious psychology.From Fig. 5~6 it will be seen that mouse enters after stomach-filling Lactobacillus brevis GDMCC No.60605
The number of open arms significantly increases, and increases 69.4%.It increased in the open arms residence time, but poor without conspicuousness with blank group ratio
It is different, illustrate that anxiety can be effectively relieved in Lactobacillus brevis GDMCC No.60605.
7 Lactobacillus brevis GDMCC No.60605 of embodiment improves sleeper effect evaluation
1 zoopery design
The 6 week old health male ICR mouses 30 for taking 20~25g, are randomly divided into 3 groups: blank control group, positive control
Group, Lactobacillus brevis GDMCC No.60605 treatment group.The daily stomach-filling sterile saline of blank control group；Lactobacillus brevis GDMCC
No.60605 treatment group feeds the concentration 3 × 10 of the preparation of this specification embodiment 1 daily9CFU/mL Lactobacillus brevis GDMCC
No.60605 degreasing emulsion suspension liquid, the diazepam solution of positive controls stomach-filling 3mg/kg.BW.Mouse phase is orally given once a day
The tested material of dosage is answered, intragastric administration on mice amount is 20mL/kg.bw, continues 30 days.Improve sleep experiments with reference to " health food is examined
With assessment technique specification (2003 editions) " progress of middle improvement sleep function value disciplines part.
1.1 extend the experiment of yellow Jackets sleeping time
First carry out preliminary experiment before doing formal experiment, determining makes animal 100% fall asleep, but do not make sleeping time it is too long penta
Barbital sodium dosage (30~60mg/kg.bw), is formally tested with this dosage.Animal last moves after giving sample 30min to each group
Yellow Jackets are injected intraperitoneally in object, and injection volume 0.2mL/20g, using righting reflex loss as index, can observation given the test agent
Extend yellow Jackets sleeping time.Experimental result is listed in attached drawing 7.
1.2 yellow Jackets sub-threshold dose hypnosis experiment
Preliminary experiment is first carried out before formal experiment, is determined pentobarbital sodium sub-threshold lull dosage (16~30mg/kg.bw), i.e.,
The yellow Jackets maximum sub-threshold dose that 80~90% mouse righting reflex do not disappear.After animal last gives sample 30min, respectively
Yellow Jackets maximum subthreshold hypnotic dosage is injected intraperitoneally in group animal, and (righting reflex loss reaches sleep number of animals in record 30min
1 minute or more person).Experiment carries out under 24~25 DEG C of quiet environments.
The experiment of 1.3 barbital sodium Sleep latencies
Preliminary experiment is first carried out before doing formal experiment, determining makes animal 100% fall asleep, but does not make sleeping time too long bar
Than the dosage (200~300mg/kg.bw) of appropriate sodium, formally tested with this dosage.After animal last awards sample 30min, to each
Barbital sodium is injected intraperitoneally in group animal, and injection volume 0.2mL/20g observes Lactobacillus brevis using righting reflex loss as index
Influence of the GDMCC No.60605 to barbital sodium Sleep latency.Experimental result is listed in attached drawing 8.
2 experimental results
2.1 extension yellow Jackets sleeping time experiments pass through preliminary experiment, it is determined that yellow Jackets intraperitoneal injection dosage
For 49mg/kg.bw.As shown in fig. 7, compared to the blank group, diazepam (3mg/kg.bw) as positive controls is extremely significant to be prolonged
The duration of yellow Jackets induced hypnotic is grown.Compared to the blank group, stomach-filling Lactobacillus brevis GDMCC No.60605's is small
Mouse sleeping time extends 149.2% and has compared significant difference with blank group, illustrates that Lactobacillus brevis GDMCC No.60605 can
Effectively to increase mouse sleep time, have the effect of improving sleep.
2.2 yellow Jackets sub-threshold dose hypnosis experiment
Influence of the 2 Lactobacillus brevis GDMCC No.60605 of table to yellow Jackets inducing mouse sleep rate
Experimental result is as shown in table 2, and after yellow Jackets (33mg/kg.bw) intraperitoneal injection of mice, positive controls are small
Mouse sleep rate is 50%, there is significant difference compared to the blank group.The mice sleep of stomach-filling Lactobacillus brevis GDMCC No.60605
Rate is 20%, increases compared to the blank group but there was no significant difference.
The experiment of 2.3 barbital sodium Sleep latencies
The dosage that barbital sodium intraperitoneal injection has been determined by preliminary experiment is 320mg/kg.bw.As shown in figure 8, and blank
Group is compared, and the incubation period of barbital sodium induced hypnotic is significantly shortened as the diazepam (3mg/kg.bw) of positive controls.With
Blank group is compared, and stomach-filling Lactobacillus brevis GDMCC No.60605 makes the Sleep latency of mouse shorten 31.0%, illustrates short cream
Bacillus GDMCC No.60605 can shorten mice sleep incubation period, it is made faster to fall asleep, and have the effect of improving sleep.
To sum up, improve sleep function value disciplines according in " health food is examined and assessment technique specification (2003 editions) "
It is required that extending the experiment of yellow Jackets sleeping time, yellow Jackets sub-threshold dose hypnosis experiment, barbital sodium Sleep latency
It is positive to test binomial in three experiments, and act on without obvious directly sleep, can determine that Lactobacillus brevis GDMCC No.60605 has
Improve sleep function effect.
Influence of the 8 Lactobacillus brevis GDMCC No.60605 of embodiment to mouse intestinal flora
1 test method
The 6 week old health male ICR mouses 30 for taking 20~25g, are randomly divided into 3 groups: blank control group, positive control
Group, Lactobacillus brevis GDMCC No.60605 treatment group.The daily stomach-filling sterile saline of blank control group；Lactobacillus brevis GDMCC
No.60605 treatment group feeds the concentration 3 × 10 of the preparation of this specification embodiment 5 daily9CFU/mL Lactobacillus brevis GDMCC
No.60605 degreasing emulsion suspension liquid, the diazepam solution of positive controls stomach-filling 3mg/kg.bw.Mouse phase is orally given once a day
The tested material of dosage is answered, intragastric administration on mice amount is 20mL/kg.bw, continues 30 days.Fresh stool in mice sample is collected, with MP excrement
Kit extracts total DNA, then using obtained bacterial genomes as template, carries out fecal specimens 16S rRNA V4 area PCR and expands
Increase, the recycling of PCR product glue, sample mixing, library construction and the sequencing of upper machine.
2 experimental results
The mouse intestinal flora beta diversity analysis analyzed by data is as shown in Figure 9, it can be seen that the short cream of stomach-filling
Conspicuousness variation has occurred in mouse flora composition after bacillus GDMCC No.60605, as can be seen from Figure 10 wherein
The relative abundance of Allobaculum dramatically increases, and most of Allobaculum are the important lifes of short chain fatty acids (SCFAs)
Production person, SCFAs is other than it can alleviate inflammation and its protection Gut barrie r and microorganism and host signal conduct most important functions
One of ingredient.So Lactobacillus brevis GDMCC No.60605 can improve sleep, may also with the relative abundance of Allobaculum
It dramatically increases, the generation for promoting SCFAs is related.
Embodiment 9: the application of Lactobacillus brevis GDMCC No.60605
(1) lactobacillus milk drink is manufactured using Lactobacillus brevis GDMCC No.60605
By raw milk defatted milk in 95 DEG C of thermal sterilization 20min, 4 DEG C are subsequently cooled to, adds Lactobacillus brevis of the invention
GDMCC No.60605 working stock culture, makes its concentration reach 106CFU/mL or more stored refrigerated is obtained containing short cream at 4 DEG C
The full bacterium milk drink of bacillus GDMCC No.60605 viable bacteria.
(2) soymilk is manufactured using Lactobacillus brevis GDMCC No.60605
Using soft water soaking soybean, water is former soybean amount three times volume, 1~2h is impregnated at 80 DEG C of temperature, then remove
Soybean skin.Then, soaking water is drained, separately adds boiling water defibrination, and keep the temperature 10~15min under conditions of temperature is higher than 80 DEG C.Slurry
Body is followed by centrifuged with 150 mesh membrane filtrations, and obtained centrifugate is thick soymilk, then heat it up temperature 140~
150 DEG C, hot thick soymilk is then imported into rapidly vacuum cooled room and is vacuumized, the smell substance in the thick soymilk is with dampening
Steam is discharged rapidly.After vacuum outgas, its temperature is down to 37 DEG C or so, then accesses Lactobacillus brevis GDMCC of the invention
No.60605 working stock culture makes its concentration reach 106CFU/mL or more stored refrigerated is obtained containing Lactobacillus brevis at 4 DEG C
The soymilk of GDMCC No.60605 viable bacteria.
(3) beverage made of fruits or vegetables is manufactured using Lactobacillus brevis GDMCC No.60605
It squeezes the juice after selecting fresh vegetables to clean, then carries out flash pasteurization, the high temperature thermal sterilization 2s at 140 DEG C of temperature
Afterwards, 37 DEG C or so are cooled to immediately, then accesses Lactobacillus brevis GDMCC No.60605 leavening of the invention, reach its concentration
106CFU/mL or more stored refrigerated obtains the beverage made of fruits or vegetables of the No.60605 viable bacteria of GDMCC containing Lactobacillus brevis at 4 DEG C.
(4) Lactobacillus brevis GDMCC No.60605 capsule product is utilized
Lactobacillus brevis GDMCC No.60605 of the invention is cultivated for 24 hours on MRS culture medium, in 4 DEG C of temperature and 4000r/
It is centrifuged 20min under conditions of min, is rinsed twice, is added to finally obtain the No.60605's of GDMCC containing Lactobacillus brevis with PBS
4% skimmed milk power of pulvis poidometer and 6% lactose mixing 10min, add sterile 2% calcium chloride and 3% sodium alginate, simultaneously
10min, then static solidification 30min are stirred with 150r/min, finally cleaning filtering, obtained filtrate carry out freeze-drying 20h, obtains
To the pulvis of the No.60605 of GDMCC containing Lactobacillus brevis, this pulvis is packed into current market sales of Development of Pharmaceutical Microcapsules, is obtained
To the capsule product.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of Lactobacillus brevis (Lactobacillus brevis) GDMCC No.60605, which is characterized in that the bacterial strain is
Guangdong Province's Culture Collection is preserved on March 12nd, 2019, preservation address is that Xianlie Middle Road, Guangzhou City is No. 100 big
5 building, the building of institute the 59th, deposit number are GDMCC No.60605.
2. Lactobacillus brevis GDMCC No.60605 according to claim 1, it is characterised in that it has the following properties:
(1) there is excellent simulated gastrointestinal tract survival ability, cell adherence ability and GABA to generate ability；
(2) mouse can be dramatically increased in spacious field experiment to enter the frequency of middle section, extend mouse in spacious field middle section
Time without influence the total move distance of mouse；
(3) number that mouse enters open arms can be dramatically increased in elevated plus-maze test；
(4) it is able to extend the sleeping time of yellow Jackets induction, shortens the Sleep latency of barbital sodium induction；
(5) intestinal flora can be adjusted, the relative abundance of Allobaculum in mouse intestinal is dramatically increased.
3. a kind of leavening, which is characterized in that contain Lactobacillus brevis GDMCC No.60605 described in claim 1.
4. leavening according to claim 3, which is characterized in that the leavening is to utilize to contain the Lactobacillus brevis
The bacterium solution of GDMCC No.60605 prepares obtained pulvis, it contains 106The active Lactobacillus brevis GDMCC of CFU/g or more
No.60605。
5. wanting any leavening of 3-4 according to right, which is characterized in that also containing in the leavening can be used for food
Microorganism.Optionally, also contain one of lactobacillus acetotolerans, lactobacillus plantarum, lactobacillus bulgaricus or two or more.
6. a kind of fermented food, which is characterized in that the fermented food is using Lactobacillus brevis GDMCC described in claim 1
What No.60605 or leavening containing Lactobacillus brevis GDMCC No.60605 strain produced.
7. fermented food according to claim 6, which is characterized in that the fermented food be dairy products, bean product or
Fruit and vegetable product.Optionally, Lactobacillus brevis GDMCC No.60605 viable bacteria is contained in the fermented food.Optionally, the cream
Product is milk, sour cream or cheese.Optionally, the bean product are soymilk, fermented soya bean or beans sauce.Optionally, the fruit
Vegetable product is cucumber, carrot, beet, celery or cabbage product.
8. Lactobacillus brevis GDMCC No.60605 described in claim 1 has the pharmaceutical composition for improving sleep function in preparation
Or the application in fermented food.
9. application according to claim 8, which is characterized in that described pharmaceutical composition is by Lactobacillus brevis GDMCC
No.60605 microbial inoculum and pharmaceutically acceptable carrier composition.
10. application according to claim 8 or claim 9, which is characterized in that pharmaceutically acceptable carrier is one or more
Carrier selected from pharmaceutically usually used filler, adhesive, wetting agent, disintegrating agent, lubricant or corrigent.It is optional
Ground, the pharmaceutical composition are granule, capsule, tablet, pill or oral liquid formulation.
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